The present invention relates to a cleaning composition comprising an acidic substantially pepstatin-insensitive protease and a nonionic surfactant. The composition is suitable for cleaning hard surfaces or cellulosic and/or woolen fabrics at acidic pH.
The majority of cleaning compositions used in household and industry today is alkaline.
Industrial cleaning like CIP (xe2x80x9cCleaning in Placexe2x80x9d) cleaning, e.g. cleaning of membranes in dairies or other food processing industries, often involves both an acid treatment whereby mineral deposits, e.g. hardness salts (scale) such as milk-stone, are removed, and an alkaline detergent treatment removing organic matter, e.g. fats, proteins and/or sugars. The process often comprises the following steps:
RINSINGxe2x86x92ALKALIxe2x86x92RINSINGxe2x86x92ACIDxe2x86x92RINSING
Lack of industrially producible acidic proteases effective at acidic pH has obstructed the development of enzymatic cleaning processes for dishes, laundry and industrial and household hard surfaces under acidic conditions, and only few attempts have been made to develop an exclusively acidic cleaning process although there is a desire for such advantageous acidic cleaning compositions.
DE 3833047 A1 discloses acidic ADW (Automatic Dish Washing) detergent compositions comprising a hydrolase enzyme, wherein the hydrolase may be an amylase, a protease or a lipase.
U.S. Pat. No. 5,698,507 discloses a gelled dishwashing composition having a pH of 3-5 consisting essentially of specified amounts of nonionic surfactant, citric acid, H2O2, at least one acid resistant protease enzyme, at least one amylase enzyme, hydrotrope, CaCl2, sodium formate, a gelling system and water. Specifically named enzymes were Bacillus amyloliquefaciens xcex1-amylases (e.g. Tenase 1200, Tenase L-1200 and Tenase L-340) and Aspergillus niger or Aspergillus oryzae proteases.
WO 95/02044 discloses acidic aspartic proteases obtainable from A. aculeatus (denoted protease I and protease II) for use in the production of food, animal feed, beverages, leather and for contact lens cleaning.
WO 96/29978 discloses an acidic oral care composition comprising acidic protease, which in the normal, slightly alkaline oral environment is substantially inactive.
WO 96/23579 discloses cleaning of membranes in a beer filtration process comprising at least a) treatment of the membrane with an enzyme-containing aqueous solution with beta-glucanase, xylanase and cellulase; b) cleaning with an acidic cleaning agent and c) cleaning with a peroxide containing alkaline solution.
We have found that proteases which retain proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, PEFABLOC, PMSF, or EDTA) exhibit a surprisingly good cleaning and/or activity performance at acidic conditions compared to other acidic proteases with a similar pH-activity profile. Accordingly, the invention provides advantages over the art of alkaline detergent compositions such as:
a) a peroxygen/activator bleach system, e.g. sodium-perborate or percarbonate and TAED activators that can oxidize or bleach poly-aromatic compounds present in soiling or stains becomes more effective even at low temperatures,
b) an enzyme-enhancer bleaching system, e.g. peroxidase-PPT or laccase-PPT may be used even at low temperatures.
The acidic condition has in itself a bleaching effect on some types of stains, e.g. coffee and tea,
c) an alkaline cleaning step and a rinsing step in industrial hard surface cleaning, e.g. CIP may be omitted as the acidic detergent composition may remove organic soils as well inorganic soil or stains,
d) omission of an alkaline cleaning step will reduce damaging of the hard surface.
e) Builder systems usually present in alkaline laundry detergents may in acidic laundry detergents be lowered or even omitted as surfactants usually are not precipitated by water hardness ions at acidic pH. This in turn means that scaling in cleaning equipment, e.g. a automatic laundry washing machine, may be avoided.
The invention thus provides in a first aspect an acidic detergent composition comprising an acidic protease which retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, PEFABLOC, PMSF, or EDTA and at least one nonionic surfactant, for hard surface and laundry cleaning.
The term xe2x80x9chard surfacexe2x80x9d as used herein relates to any surface which is essentially non-permeable to water. Examples of hard surfaces are surfaces made from metal, e.g. stainless steel or other alloys, plastics/synthetic polymers, rubber, glass, wood, concrete, rock, marble, gypsum and ceramic materials all which optionally may be coated, e.g. with paint, enamel, polymers and the like.
The term xe2x80x9cinhibitorxe2x80x9d as used herein relates to compounds which competitively or non-competitively interacts with the protease thereby reducing and/or destroying the enzyme activity towards the substrate of the enzyme.
The term xe2x80x9cretains proteolytic activityxe2x80x9d is to be construed as the protease having the property of retaining at least 75% of its activity (residual activity) measured in HPU units at pH 5.5 after treatment of the protease with inhibitor, the inhibitor being either 1 mM pepstatin, 0.1% PFABLOC, 0.1% PMSF or 100 mM EDTA.
The protease in the context of the invention is an acidic protease which retains proteolytic activity in the presence of an inhibitor selected from the group consisting of pepstatin, PEFABLOC, PMSF, or EDTA. Inhibition by pepstatin which has the formula: 
is described by Muroa S. and Oda K. (1985).
A characterization of the protease with regard to inhibition in the context of the invention is shown in WO 95/02044. An inhibition test shows that Protease I is not inhibited by pepstatin. Protease II is inhibited by pepstatin. According to Oda K. and Murao S. (1991) acidic proteases are described in two classes as carboxyl or aspartic proteases sensitive to pepstatin and pepstatin-insensitive carboxyl proteinases. Reference is also given to Muroa S. and Oda K. (1985), where this new subclass for acidic proteases was introduced.
PMSF is an inhibitor of the formula: 
while PEFABLOC is a protease inhibitor of the formula: 
Preferred proteases are obtainable from a microorganism, e.g. a bacterial strain, e.g. Bacillus, Pseudomonas or Xanthomonas or a fungal strain (including yeasts) such as species of the genus Aspergillus (e.g. A. aculeatus or A. niger) or Scytalidium (e.g. S. lignicolum).
The proteases may in a further embodiment be obtainable from a bacterium or fungus, which has been genetically modified by transforming said bacterium or fungus with a DNA vector/construct comprising DNA encoding said protease.
The protease of the invention may, in a particularly preferred embodiment, comprise one or more aspartic and/or carboxylic residues as functional groups in the active center.
Further, the protease retains proteolytic activity in the presence of inhibitors present in meat, egg white, whole blood, blood plasma, milk, beer, potatoes or beans. Such inhibitors may be ovomacroglobulin, ovomucoid or ovoglycoprotein. It is presently contemplated that inhibitors (such as competitive or non-competitive inhibitorsxe2x80x94terms that are known to the skilled person) present in soiling which is desired to be cleaned play an important role in the cleaning process as they may inactivate or reduce activity of enzymes which would otherwise hydrolyze the soiling. This may be the reason why it has been difficult to find suitable proteases for use in acidic cleaning compositions. The protease may further have a preferred pH optimum between 2-7, more preferred between 3-6 or even more preferred between 4.5-5.5. Also the protease according to the invention may have a temperature optimum between 20-70xc2x0 C., such as between 20-60xc2x0 C., e.g. 30-50xc2x0 C.
In a further particular embodiment the protease is Protease I or Protease II obtainable from A. aculeatus as described in WO 95/02044, which is hereby incorporated by reference. A most preferred protease is Protease I.
Nonionic surfactants are especially suitable for acidic detergents since they are not functionally affected in a moderate acidic environment. Preferred nonionic surfactants are glycolipids, alcoholethoxylates, alkylphenolethoxylates, glucamides, and alkylpolyglucosides (Stache and Kosswig, 1990).
The composition may optionally comprise a sequestering agent, when water for preparing a washing liquor contains considerable water hardness, i.e. calcium ions which may affect the performance of the protease. Suitable sequestering agents should be capable of sequestering calcium ion at acidic pH. Preferred sequestering agents are methylglycinediacetic acid, nitrilotriacetic acid, citric acid, oligo and polymeric (poly)carboxylic acid derived from polymer sugars (Kock et al. 1993), dextrin and protein hydrolysates (DE 19547730 A1).
The composition may further comprise other components enhancing the detergency of the composition such as softening agents, an amylase (e.g. Fungamyl(copyright) from Novo Nordisk A/S, Denmark), a lipase (e.g. Novocor(copyright) AD from Novo Nordisk A/S, Denmark), a cellulase (e.g. Celluzyme(copyright), Carezyme(copyright), and/or Celluclast(copyright), all from Novo Nordisk A/S, Denmark), a xylanase (e.g. Biofeed(copyright) PLUS or Shearzyme(trademark) from Novo Nordisk A/S, Denmark), a beta-glucanase (e.g. Viscozyme(copyright) or Ultraflo(trademark) from Novo Nordisk A/S, Denmark), a pectinase (e.g. Pectinex(trademark) Ultra from Novo Nordisk A/S, Denmark), a peroxidase (e.g. Guardzyme(trademark) from Novo Nordisk A/S, Denmark), a laccase (e.g. obtained from Myceliophthora or Polyporus), an enhancing agent for the peroxidase/laccase (e.g. PPT or methylsyringic acid methylsyringate or derivatives thereof) and/or a buffer (e.g. citric acid).
Finally the composition may be a liquid or a powder. In the latter case the protease may suitably be formulated as a stabilized granulate. The formulation may be obtained using conventional methods.
The composition according to the invention may suitably be used in methods for cleaning or washing a hard surface or laundry. The methods may preferably comprise contacting the hard surface or laundry with the composition dissolved in an aqueous solution in an amount sufficient for providing a cleaning effect. The protease and the other composition components may alternatively be added to the solution separately.
The composition may preferably be dissolved in an amount sufficient for providing an enzyme dosage of 500-3000 HUT/L, preferably 500-1500 HUT/L, more preferably 750-1250 HUT/L, e.g. 1000 HUT/L wash liquor.
The hard surface to be cleaned or washed by the method is in one embodiment preferably industrial process equipment or household equipment.
Specific preferred industrial process equipment may be heat exchangers, tanks, pipes, centrifuges, evaporators, filters, extruders, meat choppers, cooking jars, beer and wine fermenters, beer and wine filters, spent filter aids, coolers, storage tanks, sieves, hydrocyclones, ultrafiltration units, nanofiltration units, hyperfiltration units, microfiltration units and milking machines.
A particularly suitable embodiment is cleaning of health care or animal care equipment or products. Health care equipment may comprise diagnostic/analytical (e.g. endoscopes, blood analyzers), processing (e.g. dialysis or blood treatment equipment) or surgical equipment (e.g. scalpels, peens, clips or tweezers, forceps, etc. used by doctors, veterinarians or dentists for treatment of patients) which has been in contact with blood, other body fluids or tissues from humans or animals.
With respect to cleaning of industrial process equipment the methods may in particular embodiments be xe2x80x9cCleaning in Placexe2x80x9d (CIP) methods.
Specific preferred household equipment may be eating utensils, plates, cups, beakers, glasses, pots, pans, electric appliances, toilet bowls, lavatories or tiles.
Another suitable embodiment of the invention is cleaning of industrial ion exchange columns used in bulk sugar production for removing salts, residues of charged carbohydrates, protein and amino acids, and colored material before crystallization. Furthermore, ion exchange columns used in the starch based syrup production before and after the isomerization process may be cleaned using the composition of the invention.
An additional preferred embodiment of the invention is cleaning of ion exchangers used in manufacturing processes where proteins or peptides may cover and/or clog up the resin material with proteinaceous material. Usually such clogged up ion exchanges needs a cleaning process based on excessive alkaline and heat conditions, which reduces the lifetime of the resin and reduces the efficiency of the regeneration process (e.g. by using 85xc2x0 C. hot alkali at pH=13-14 recirculated for about an hour). Use of the composition of the invention, however, provides a mild cleaning process, which may secure an efficient removing of soil that hinders an efficient regeneration process of such ion exchangers.
Yet another particularly suitable embodiment of the invention is cleaning of columns used for protein separation by gel filtration or affinity chromatography. Such columns often contain rather expensive separation and/or chromatography material/resin, which needs to be cleaned effectively if it is used for scaled up processes. Harsh conditions are normally used for removing slimy material such as combination products of protein and carbohydrates, which deposits in the material and these harsh conditions often reduce the lifetime of the material. Use of the composition of the invention, however, provides a mild cleaning process, which may secure an efficient cleaning and a prolonged lifetime of the column material. A preferred cleaning process for cleaning columns containing gel filtration and affinity chromatography material may comprise recirculating a cleaning solution containing an enzyme dosage of 500-3000 HUT/L, preferably 500-1500 HUT/L, more preferably 750-1250 HUT/L, e.g. 1000 HUT/L wash liquor at a pH between 2-7, preferably 3-6, e.g. 4-5, while the temperature should be kept between 10-65xc2x0 C., preferably 30-50xc2x0 C., e.g. 40xc2x0 C. The cleaning or washing time in a preferred embodiment is kept between 2 minutes and 20 hours depending on the type of method.
With respect to cleaning of household equipment, the methods may in particular embodiments be performed in an automatic dishwashing machine.
When using the composition for cleaning or washing of laundry the method may preferably be performed in an industrial scale or household scale washing machine. Preferred laundry is cellulosic fabric and/or non-structured garments such as silk, acetate, wool, ramie, or rayon garments. Cleaning of especially wool and silk according to the invention is useful, as acidic cleaning conditions softens the garments as well as having a antimicrobial effect (i.e. kills or inhibits microbial cells).
The methods for cleaning or washing of hard surfaces or laundry may in one embodiment be performed at a pH between 2-7, preferably 3-6, e.g. 4-5, while the temperature should be kept between 10-65xc2x0 C., preferably 30-50xc2x0 C., e.g. 40xc2x0 C. The cleaning or washing time in a preferred embodiment is kept between 2 minutes and 20 hours depending on the type of method. For instance cleaning of an industrial membrane may provide soaking in a (circulating) solution of the composition for up to overnight (up to 20 hours), while industrial dishwashing should be completed within 2-10 minutes.
The HUT activity was determined according to the AF92/2 method published by Novo Nordisk A/S, Denmark. 1 HUT is the amount of enzyme which, at 40xc2x0 C. and pH 4.7 over 30 minutes, forms a hydrolysate from digesting denatured hemoglobin equivalent in absorbancy at 275 nm to a solution of 1.10 xcexcg/ml tyrosine in 0.006 N HCl which absorbancy is 0.0084. The denatured hemoglobin substrate is digested by the enzyme in a 0.5 M acetate buffer at the given conditions. Undigested hemoglobin is precipitated with trichloroacetic acid and the absorbance at 275 nm is measured of the hydrolysate in the supernatant.
1 hemoglobin protease unit (hpu) is defined as the amount of enzyme liberating 1 millimole of primary amino groups (determined by comparison with a serine standard) per minute under standard conditions as descried below:
A 2% (w/v) solution of hemoglobin (bovine, supplied by Sigma) is prepared with the Universal Buffer described by Britton and Robinson, J. Chem. Soc., 1931, p. 1451), adjusted to a pH of 5.5. 2 ml of the substrate solution are pre-incubated in a water bath for 10 min. at 25xc2x0 C. 1 ml of an enzyme solution containing b g/ml of the enzyme preparation, corresponding to about 0.2-0.3 hpu/ml of the Universal Buffer (pH 5.5) is added. After 30 min. of incubation at 25xc2x0 C., the reaction is terminated by the addition of a quenching agent (5 ml of a solution containing 17.9 g of trichloroacetic acid, 29.9 g of sodium acetate and 19.8 g of acetic acid made up to 500 ml with deionized water). A blank is prepared in the same way as the test solution with the exception that the quenching agent is added prior to the enzyme solution. The reaction mixtures are kept for 20 min. in a water bath after which they are filtered through Whatman 42 paper filters.
Primary amino groups are determined by their color development with o-phthaldialdehyde (OPA), as follows: 7.62 g of disodium tetraborate decahydrate and 2.0 g of sodium dodecylsulfate are dissolved in 150 ml of water. 160 mg of OPA dissolved in 4 ml of methanol were then added together with 400 ml of xcex2-mercaptoethanol after which the solution is made up to 200 ml with water. To 3 ml of the OPA reagent are added 400 ml of the filtrates obtained above, with mixing. The optical density (OD) at 340 nm is measured after about 5 min. The OPA test is also performed with a serine standard containing 10 mg of serine in 100 ml of Universal Buffer (pH 5.5). The buffer alone is used as a blank. The protease activity is calculated from the OD measurements by means of the following formula:       hpu    /    ml    ⁢      xe2x80x83    ⁢  enzyme  ⁢      xe2x80x83    ⁢  solution  ⁢      :      ⁢                    (                              OD            t                    -                      OD            b                          )            xc3x97              C        SER            xc3x97      Q                      (                              OD            SER                    -                      OD            B                          )            xc3x97      M      ⁢              xe2x80x83            ⁢              W        SER            xc3x97              t        i            
hpu/g of enzyme preparation=hpu/ml: b
wherein ODt, ODb, ODSER and ODB is the optical density of the test solution, blank, serine standard and buffer, respectively, CSER is the concentration of serine (mg/ml) in the standard (in this case 0.1 mg/ml), and MWSER is the molecular weight of serine (105.09). Q is the dilution factor for the enzyme solution (in this case 8) and ti is the incubation time in minutes (in this case 30 minutes).
Stainless steel plates and porcelain dishes had been pre-washed by 75xc2x0 C. at high alkaline pH before soiling. Efficiency of proteases were tested using a standard laboratory procedure where stainless steel plates were soiled in the following way: 15 eggs (white+yolk) and 255 ml of whole milk were mixed at the lowest speed in a Braun UK 20 kitchen mixer machine for 2 minutes. Thereafter the mixed solution passed through a 0.5 mm pore size sieve. Five stainless steel plates were dipped into the egg/milk mixture and placed in a drying rack. After drying overnight at room temperature, the plates were baked for 1 hour at 120xc2x0 C. in a ventilated thermostated oven. To test amylases, five porcelain plates are soiled using a 2% suspension of gelatinized starch solution which is dried overnight at room temperature.
For the automatic dishwashing procedure, a 6 persons Cylinda Excellence Kompakt type 770 machine was used. The machine had an ion-exchanger installed binding calcium ions from the inlet water. Temperature was set to 55xc2x0 C. at program no. 4. This program performed the following:
a) Main wash starting with 7 minutes of heating of the cleaning solution from 25xc2x0 C. to 55xc2x0 C. and ending with 10 minutes of washing.
b) Cold rinsing water is taken in. This rinsing takes 5 minutes and the temperature during rinsing varies between 35 and 45xc2x0 C. dependent on loading.
c) A second rinsing made with cold water which is taken in. During the second rinsing the water is heated from 20xc2x0 C. to 55xc2x0 C. for 8 minutes and then pumped out.
d) Drying at 55xc2x0 C. for about 5 minutes.
Before and after washing, light reflection values are measured directly for protein film or for starch films stained with iodine (KI/I2). The staining was made using a standard solution which was prepared the following way: 20.0 grams of potassium iodide (KI) Merck art. No. 5043 and 1.27 gram of iodine (I2) Merck art. No. 4763 were weighed out in a 2 liter beaker and added up to 1.0 liter using ion exchanged water. The solution was stirred for approximately 10 minutes at room temperature.
The plates were pulled slowly through the iodine solution and afterwards placed in drying racks.
Measurements are made using a Minolta Chroma Meter (type CR-300). Six single values for each plate are used for an average R-value. Calculations of % removed filmxe2x80x94RPF% for protein or RSF% for starchxe2x80x94are calculated by the formula:
RPF(%) [or RSF(%)]=[Rafter washxe2x88x92Rbefore wash]/[Rclean platexe2x88x92Rbefore wash]*100%
Detergent composition used for one machine and the cleaning results obtained are shown in each of the tables belonging to the examples described below.
pH was varied by addition of 2-7 ml of 4 N HCl.
Test of washing performance was carried out in a commercial European washing machine (AEG model xc3x96KO-LAVAMAT JUBILEUM 40) with 2.0 kg of ballast laundry and artificially soiled fabrics. 10 pieces (5xc3x975 cm) of a commercial xe2x80x9cstandardxe2x80x9d swatch (standard cotton fabric) soiled with milk, blood and carbon black (EMPA 116) and 10 pieces of a swatch impregnated with an extract of spinach leaves and later on heat treated at 70xc2x0 C. for 30 minutes were fixed onto the ballast cloth. The washing process was performed at 40xc2x0 C. without pre-wash using a program called xe2x80x9cKlarvaskxe2x80x9d according to the instructions by the vendor, with a final centrifugation at 1400 rpm. During the washing process a sample of the washing liquor was taken and the pH was measured using a pH-indicator strip type Acilit(copyright) pH 0-6. The washing process was followed by 2 rinsings with intermediate centrifugations at 400 rpm. In the total process a total volume of 50 liters of water were used.
Assessment of washed test swatches was done by measuring the intensity of reflected light, % R, (% remission) remitted from the swatches at 460 nm using a JandM Tidas MMS/16 photometer equipped with a CLX 75W Xenon lamp and fiber optics. Each swatch was measured individually at the top of a stack with 3 or 4 other swatches (in order to diminish the amount of light which may penetrate the textile structure without being absorbed or reflected).
The value xcex94REnz=Rwashedxe2x88x92Rwashed without enzyme reflects the contribution of the enzyme for each type of swatch. The results are illustrated as mean values and as confidence intervals, e.g. as [% Rwashedxe2x88x92W; % Rwashed+W], where W is the 95% confidence value.
a) NTA (nitrilotriacetic acid) available from Fluka Chemika no. 72560).
b) Trilon(copyright) A (NTA-Na3xe2x80x94nitrilotriacetic acid trisodium salt) available from BASF - Germany.
c) Trilon(copyright) M (MGDA-Na3xe2x80x94methylglycinediacetic acid trisodium salt) available from BASF - Germany
d) Dehyphon(copyright) LS 54 (a non-ionic alcoholethoxylate) available from Henkel KGaA - Germany
e) Lutensol(copyright) AO3 (a non-ionic alcoholethoxylate) available from BASF - Germany
f) Lutensol(copyright) AO 7(a non-ionic alcoholethoxylate) available from BASF - Germany)
g) Sokalan(copyright) HP25 (a modified polycarboxylate) available from BASF - Germany; used as anti-redeposition agent.
h) Protease I (1.05 kHUT/g) obtained as described in WO 95/02044.
i) Protease II (5.22 kHUT/g) obtained as described in WO 95/02044.
j) Flavourzyme (65.2 kHUT/g) available from Novo Nordisk A/S, Denmark.
k) Fungamyl(copyright) available from Novo Nordisk A/S, Denmark.